Antimicrobial peptide response to group B Streptococcus in human extraplacental membranes in culture.

OBJECTIVE: Streptococcus agalactiae (GBS) is an important cause of chorioamnionitis. This study characterizes GBS colonization and stimulation of antimicrobial responses in human extraplacental membranes using an ex vivo transwell two-compartment system of full-thickness membranes and live GBS. STUDY DESIGN: Human extraplacental membranes were affixed to transwell frames (without synthetic membranes). Live GBS was added to the decidual side of membranes in transwell cultures, and cocultures were incubated for 4, 8 and 24 h. GBS recovery from homogenized membranes and culture medium was determined by enumerating colony forming units (CFU) on blood agar. Antimicrobial peptide expression was identified using immunohistochemistry and ELISA. GBS killing by HBDs was assessed in vitro by incubating GBS with different human beta defensins (HBDs) for 3 h, then enumerating CFU. RESULTS: GBS recovery from membranes markedly decreased over time (P < 0.05). The antimicrobial peptides HBD-1, HBD-2, HBD-3, and lactoferrin were expressed in both GBS-exposed and non-exposed tissues. Notably, a pattern of localized increased HBD-2 in the amnion of GBS-infected tissue was observed. Moreover, GBS-treated membranes released increased amounts of HBD-2 into the amniotic and decidual compartments of the transwell cultures after 24 h (P < 0.05). In bacterial cultures, HBD-2 decreased GBS viability in a concentration-dependent manner (P < 0.05). CONCLUSION: Innate immune responses in ex vivo human extraplacental membranes suppress GBS growth. HBD-2 was implicated in this GBS suppression with evidence of signal transduction across the tissue. Antimicrobial peptides may be important for innate immune defense against intrauterine GBS infections during pregnancy.

Cell and toxicant specific phosphorylation of conexin43: effects of lindane and TPA on rat myometrial and WB-F344 liver cell gap junctions.

Previous studies showed that the pesticide lindane (gamma-hexachlorocyclohexane) inhibits gap junction intercellular communication in rat myometrial cells. The present study tested the hypothesis that lindane and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibit gap junction communication in rat myometrial and liver WBr-F344 cells by the common mechanism of increasing phosphorylation of the gap junction protein connexin43. We evaluated changes of connexin43 phosphorylation using Western blot of standard SDS-PAGE gels and cell immunostaining, and we monitored gap junction communication using microinjection and transfer of Lucifer yellow dye. Exposure of rat myometrial cells to lindane or TPA nearly abolished dye transfer but did not alter the electrophoretic mobility of connexin43, and neither lindane nor TPA increased phosphorylation of connexin43 as assessed by immunoblot with anti-phospho-connexin43 (S368) antibody. However, TPA increased punctate immunofluorescence staining of phospho-connexin43 (S368) in myometrial cells whereas lindane had no such effect. In WBr-F344 cells, lindane and TPA inhibited dye transfer. Lindane increased immunostaining for phospho-connexin43 (S368) in WBr-F344 cells without altering the abundance, electrophoretic mobility or phosphorylation of connexin43 as detected in immunoblots. TPA intensified a slower migrating connexin43 band and increased phospho-connexin43 (S368) in immunoblots, and intensified phospho-connexin43 immunostaining at WBr-F344 cell interfaces and nuclear regions. These results show that phosphorylation of connexin43 at serine 368 occurred in cell and toxicant specific manners and was independent of changes in electrophoretic mobility in standard SDS-PAGE gels. Moreover, lindane inhibited gap junction communication in myometrial cells by a mechanism that was not be explained by changes in phosphorylation of connexin43.

Stimulation of contraction of pregnant rat uterus in vitro by non-dechlorinated and microbially dechlorinated mixtures of polychlorinated biphenyls.

A previous study of six polychlorinated biphenyl (PCB) congeners showed that PCBs with four or fewer chlorines and ortho substitution stimulate uterine contraction frequency in vitro, whereas congeners with a greater number of chlorines or non-ortho substitution are inactive in vitro. We tested the hypothesis that PCB mixtures stimulate uterine contractions in a manner inversely related to the degree of chlorination and the presence of chlorines in the ortho- position of the biphenyl constituents of the mixtures. Uterine strips from pregnant rats were suspended in standard muscle baths and analyzed for changes in isometric contractions in response to in vitro exposure to commercial PCB mixtures (Aroclors) and their dechlorinated products after microbial degradation. The PCB mixtures Aroclor 1242, 1248, and 1254 significantly stimulated uterine contraction frequency, and the least chlorinated mixture, Aroclor 1242, was the most potent stimulant. Microbes from Hudson River sediment dechlorinated Aroclor 1242 and Aroclor 1254 under reducing conditions to produce mixtures with an increased proportion of ortho-substituted congeners with one or two chlorine substitutions. The PCB mixtures that had undergone microbial reductive dechlorination stimulated uterine contraction frequency to a significantly greater extent than the parent mixtures. These results show that increased uterotonic activity was associated with decreased chlorination and increased ortho substitution of the biphenyl constituents of the mixtures.

Antioxidants prevent gamma-hexachlorocyclohexane-induced inhibition of rat myometrial gap junctions and contractions.

Lindane (gamma-hexachlorocyclohexane) is a commonly used pesticide that bioaccumulates in mammalian adipose tissue. Lindane inhibits gap junctional intercellular communication and oscillatory contractions of pregnant rat myometrium in vitro. The present study investigated the role of oxidative stress in lindane's inhibition of myometrial function in mid-gestation pregnant rat uteri. Lucifer yellow dye was microinjected into cultured myocytes to assess gap junctional intercellular communication. Lindane exposure (100 microM) resulted in a time-dependent, biphasic inhibition of dye transfer. This pattern of inhibition was also seen upon cell exposure to the pro-oxidant, tert-butyl hydroperoxide (100 microM). Lindane's initial and secondary-onset dye transfer inhibitions were reversed by cotreatment and pretreatment with the antioxidants, alpha-tocopherol (25-100 microM), diphenyl-1,4-phenylene diamine (10-30 microM), and superoxide dismutase (100-400 U/ml). D-mannitol (100-300 mM) also reversed lindane's initial dye transfer inhibition. Nitro blue tetrazolium reduction to formazan (measured spectrophotometrically) was elevated upon exposure of cultured cells to lindane or tert-butyl hydroperoxide, indicating the presence of reducing agents. Lipid peroxidation, assessed as thiobarbituric acid-reactive substances, was also elevated in lindane-exposed cell cultures. alpha-Tocopherol reversed this elevation. Finally, uterine contractility was assessed by measuring isometric contractions of uterine strips hung in standard muscle baths. Pretreatment with alpha-tocopherol prevented lindane's abolishment of uterine contractions in vitro. These data support the hypothesis that lindane inhibits uterine contractility and myometrial gap junctions by establishing an oxidative stress environment.

A calcium-independent phospholipase activity insensitive to bromoenol lactone mediates arachidonic acid release by lindane in rat myometrial cells.

Arachidonic acid release is an important regulatory component of uterine contraction and parturition, and previous studies showed that lindane stimulates arachidonic acid release from myometrium. The present study partially characterized the enzyme activity responsible for lindane-induced arachidonic acid release in myometrial cells. Lindane released arachidonic acid from cultured rat myometrial cells in concentration- and time-dependent manners. This release was primarily from phosphatidylcholine and phosphatidylinositol, and was independent of intracellular and extracellular calcium. In cells prelabeled with [3H]arachidonic acid, 85% of radiolabel was recovered as free arachidonate and only 5% was recovered as eicosanoids. Pretreatment with the antioxidants Cu, Zn-superoxide dismutase, alpha-tocopherol or Trolox did not significantly modify lindane-induced arachidonic acid release. Pretreatment of cells with the phosphatidylcholine-specific phospholipase C inhibitor D609, phosphatidylinositol-specific phospholipase C inhibitor ET-18-OCH3, or an interrupter of the phospholipase D pathway (ethanol) did not suppress lindane-induced arachidonic acid release. Although these results are consistent with calcium-independent phospholipase A2 activation by lindane, the calcium-independent phospholipase A2 inhibitor bromoenol lactone failed to inhibit lindane-induced arachidonic acid release in myometrial cells, even though bromoenol lactone effectively blocked arachidonic acid release in neutrophils. These results suggest that myometrial cells express a novel, previously unidentified phospholipase that is arachidonate-specific, calcium-independent, insensitive to bromoenol lactone, insensitive to reactive oxygen species activation, shows substrate preference for phosphatidylcholine and phosphatidylinositol, and is stimulated by lindane. Moreover, the data show that the overwhelming majority of arachidonic acid released remains as arachidonate, but that lindane does not significantly inhibit metabolism of arachidonate to eicosanoids.

Lindane-induced inhibition of spontaneous contractions of pregnant rat uterus.

Hexachlorocyclohexanes (HCHs) are prevalent insecticides. Lindane (gamma-HCH) inhibits uterine gap junctions but beta-HCH does not. Because gap junctions promote coordination of oscillatory uterine contractions, we hypothesized that lindane, but not beta-HCH, would inhibit uterine contractions. Uterine strips from midgestation rats were suspended in standard muscle baths and exposed to HCHs in a cumulative manner. Lindane induced concentration-dependent decreases in contraction force (ED50 of 9.2 microM) and complete uterine quiescence at 30 microM. In contrast, beta-HCH had no effect on contraction force, but 20 to 200 microM beta-HCH increased contraction frequency in a concentration-dependent manner. Isomer-specific differences in uterine responses were observed at similar HCH isomer tissue concentrations. Additionally, the phospholipase A2 inhibitor and antioxidant quinacrine increased the ED50 for contraction force inhibition to 84.5 microM lindane. Lindane also increased cAMP concentrations. Lindane and beta-HCH have distinctly different actions in the uterus. Lindane's inhibitory action may involve cAMP, arachidonic acid, or oxidative stress.

Stimulation of pregnant rat uterine contraction by the polychlorinated biphenyl (PCB) mixture aroclor 1242 may be mediated by arachidonic acid release through activation of phospholipase A2 enzymes.

The polychlorinated biphenyl (PCB) mixture Aroclor 1242 (A1242) increases frequency of contractions of pregnant rat uteri, suggesting a possible mechanism for decreased gestational age and increased spontaneous abortion in women and animals exposed to PCBs. In the present study, we hypothesized that A1242-induced stimulation of uterine contraction is mediated by arachidonic acid released by phospholipase A2 (PLA2) enzymes. Isometric uterine contraction was measured in longitudinal uterine strips isolated from gestation day 10 rat. Pretreatment of uterine strips with the PLA2 inhibitor (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (HELSS) or manoalide, or an inhibitor of the G protein of PLA2, isotetrandrine, completely prevented the increase of contractile frequency induced by 50 microM A1242. However, the phospholipase C inhibitors 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC) and neomycin were unable to block stimulation of uterine contraction by A1242. In accordance, A1242 (100 microM) did not release inositol phosphates from myo-[3H]inositol-labeled myometrial cells, whereas myometrial cells prelabeled with [3H]arachidonic acid released arachidonic acid in a concentration- and time-dependent manner after exposure to A1242 (10-100 microM). A1242 significantly stimulated arachidonic acid release in the absence of extracellular calcium, although the release was attenuated. Analysis of the eicosanoids released by A1242 indicated that only 0.83% of released [3H]arachidonic acid was metabolized to eicosanoids and 99.07% remained as free arachidonate. Uterine contraction increased in strips exposed to exogenous arachidonic acid (1-100 microM). This study suggests that A1242 stimulates contraction in pregnant rat uterus by a mechanism involving PLA2-mediated arachidonic acid release, and that arachidonic acid, rather than eicosanoids, may mediate A1242 uterotonic action in the uterus.

A mechanistic-based approach for assessing chemical hazards to parturition.

Evaluations of environmental hazards to pregnancy often overlook the potential for chemicals to disrupt the final event, childbirth. There are relatively few epidemiologic studies on this topic and even fewer toxicologic investigations. Mechanistic-based approaches offset many of the difficulties that are anticipated with intact laboratory animals, such as interspecies variability in the initiating events, and may allow for rapid and relevant assessment of potential chemical hazards. In vitro systems based on knowledge of the cellular events that underlie parturition may, therefore, facilitate investigation of toxicologic aspects of parturition. Nonetheless, limitations of in vitro mechanistic-based approaches exist. Ultimately, the greatest understanding of risk to pregnancy from environmental chemicals is likely to result from the collaborative efforts of laboratory scientists and epidemiologists.

Stimulation of oscillatory uterine contraction by the PCB mixture Aroclor 1242 may involve increased [Ca2+]i through voltage-operated calcium channels.

Polychlorinated biphenyls (PCBs) are persistent environmental pollutants associated with spontaneous abortion and shortened gestation length in women and animals. In previous studies, we showed that PCB mixtures and noncoplanar ortho-substituted PCB congeners increased contractions in pregnant rat uterus. In the present study, we hypothesized that the PCB mixture Aroclor 1242 (A1242) stimulates oscillatory uterine contraction in pregnant uterus by increasing intracellular calcium concentration ([Ca2+]i). Pretreatment of uterine strips with ryanodine or thapsigargin, to deplete specific intracellular calcium stores, did not prevent the increased frequency of oscillatory contraction due to 50 microM A1242, whereas thapsigargin effectively blocked carbachol-induced stimulation of uterine contraction. However, 100 microM A1242 was unable to increase contraction in the absence of extracellular calcium or in the presence of the voltage-operated L-type calcium channel blocker nifedipine. A1242 (100 microM) was observed to partially depolarize the cell membrane of myometrial cells from pregnant rats, as measured with a potential-sensitive carbocyanine dye. Changes of [Ca2+]i were monitored in single myometrial cells loaded with the fluorescent calcium-sensitive probe fura-2. Cells exposed to 100 microM A1242 showed a delayed and sustained increase of [Ca2+]i, and this increase was completely blocked in the absence of extracellular calcium or the presence of nifedipine. Therefore, the data suggest that depolarization of the cell membrane by A1242 enabled myometrial cells to increase [Ca2+]i through activation of voltage-operated calcium channels, and the increased [Ca2+]i consequently stimulated contraction of uterine smooth muscle.

Acute inhibition of spontaneous uterine contractions by an estrogenic polychlorinated biphenyl is associated with disruption of gap junctional communication.

An estrogenic polychlorinated biphenyl, 4-hydroxy-2',4', 6'-trichlorobiphenyl (4-OH-TCB), inhibits oscillatory uterine contractions immediately. Because increased gap junction formation is associated with the development of synchronized uterine contractions at term, we examined whether the inhibitory effect of 4-OH-TCB on spontaneous oscillatory contractions was due to the disruption of gap junctional communication. The effect of 4-OH-TCB on gap junctional communication was determined by intercellular Lucifer yellow dye transfer in primary cultures of myometrial myocytes isolated from midgestation rats. Intercellular dye transfer was inhibited by 4-OH-TCB or 17beta-estradiol in a concentration-dependent manner. The inhibitory effect of 4-OH-TCB on intercellular dye transfer was reversed by tetraethylammonium (TEA). To examine effects on uterine contraction, longitudinal uterine strips were excised from midgestation rats and placed in muscle baths for isometric force measurement. Spontaneous uterine oscillation was suppressed by 4-OH-TCB or 17beta-estradiol. The inhibitory effects of 4-OH-TCB and 17beta-estradiol on spontaneous oscillations were counteracted by TEA but were not affected by a calcium ionophore (A23187) or a calcium-dependent potassium channel blocker (apamin). These results suggest that the acute inhibition of spontaneous oscillatory contractions by an estrogenic polychlorinated biphenyl may result from the disruption of intercellular communication.

2-Methoxyethanol inhibits gap junctional communication in rat myometrial myocytes.

The glycol ethers 2-methoxyethanol (2-ME) and 2-ethoxyethanol (2-EE) prolong gestation in rodents. Because gap junctions in the myometrium likely facilitate parturition, the present study examined inhibition of gap junctional communication by 2-ME and 2-EE in myometrial smooth-muscle cell cultures. To measure gap junctional communication, the fluorescent dye Lucifer yellow was injected into cultured cells and the transfer of the dye to adjacent cells was scored with epifluorescence microscopy. The data are presented as the percentage of cells adjacent to the microinjected cell that exhibited dye following microinjection. A 30 min treatment with 32 or 63 mmol/L 2-ME decreased dye transfer to 71% and 63%, respectively (p < or = 0.05; control 90%). Similarly, 2-EE inhibited dye transfer, although myometrial cells were less sensitive to 2-EE compared to 2-ME. Dye transfer returned to control levels after 2 h in the continued presence of 2-ME. The primary metabolite of 2-ME, methoxyacetic acid (MAA), had no effect on dye transfer at concentrations equimolar to 2-ME. Because 2-ME and 2-EE inhibited gap junctional communication only at high concentrations and because the inhibition reversed in the continued presence of the compounds, it is suggested that glycol ethers delay parturition by a mechanism independent of a direct action on myometrial gap junctions.

Increase of oxytocin-induced oscillatory contractions by 4-hydroxy-2',4',6'-trichlorobiphenyl is estrogen receptor mediated.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants that are associated with decreased gestation length in women as well as other mammals. Many lightly chlorinated PCBs are hydroxylated in vivo. The PCB congener 4-hydroxy-2',4',6'-trichlorobiphenyl (4-OH-TCB) has a high affinity for estrogen receptors and exerts a uterotropic effect in vivo. This study tested the hypothesis that 4-OH-TCB increases the contractile response of midgestation uteri to oxytocin by an estrogen receptor-mediated mechanism. After in vitro treatments with 4-OH-TCB or estradiol-17beta for 20 h or 42 h, uterine explants from midgestation rats were mounted in standard muscle baths for measurement of isometric contractions. A 20-h exposure to either 4-OH-TCB (0.1, 1, or 10 microM) or estradiol-17beta (10 nM) failed to alter the contractile response to cumulative additions of oxytocin (10(-10) to 10(-7) M). However, a 42-h exposure to either 1 microM 4-OH-TCB or 10 nM estradiol-17beta significantly elevated the contractile response to oxytocin, which was abolished by cotreatment with the estrogen receptor antagonist tamoxifen (30 nM). These data support the hypothesis that the stimulatory actions of estradiol-17beta and 4-OH-TCB on oxytocin-induced oscillatory contractions are mediated by estrogen receptors. Under the conditions of this experiment, more than 20 h of treatment is required to elicit the estrogen-dependent responses.

Congener-specific effects of PCBs on contractions of pregnant rat uteri.

Both increased and decreased gestation lengths have been reported following exposures to polychlorinated biphenyl (PCB) mixtures and congeners. Because oscillatory uterine contractions are essential for parturition, we hypothesized that the disparate findings on gestation length may be the result of distinct PCB congener-specific actions on oscillatory uterine contractions. This study examined the acute effects of PCB congeners on isometric contractions of isolated pregnant uteri and the structure-activity relationship for individual congeners. After cumulative exposure to individual PCB congeners (0.5 microM to 150 microM), oscillatory contractions were: 1) not altered by 2,4,5,2',4',5'-hexachlorobiphenyl, 3,4,5,3',4'-pentachlorobiphenyl, or 3,4,3',4'-tetrachlorobiphenyl; 2) significantly inhibited by 4-hydroxy-2',4',6'-trichlorobiphenyl; and 3) markedly increased by 2,4,6-trichlorobiphenyl and 2,4,2',4'-tetrachlorobiphenyl, when compared to solvent controls. The uteri were more sensitive to PCB congeners with ortho-substituted light chlorination than those highly chlorinated, or those interacting with the Ah-receptor.

Lindane-induced elimination of gap junctional communication in rat uterine myocytes is mediated by an arachidonic acid-sensitive cAMP-independent mechanism.

Previous studies by this laboratory showed that the pesticide lindane rapidly and potently inhibits gap junctional communication in myometrial smooth muscle cells. This study examined the possible role of cAMP or arachidonic acid in lindane's elimination of myometrial gap junctional communication. Lindane produced concentration-dependent increases in cAMP of 1.21, 2.94, 6.06, and 8.69 pmol/mg protein with 0.1, 1, 30, and 100 microM lindane, respectively, compared to solvent-treated controls (1.27 pmol/mg protein). Lindane also increased release of tritiated arachidonic acid to 342, 509, 852, 1236, 1639, and 4454 dpm/micrograms protein with 0.01, 0.1, 1, 10, and 100 microM lindane, respectively, compared to solvent controls (342 dpm/micrograms protein). Transfer of Lucifer Yellow dye was used as a measure of gap junctional communication. Both 8-br-cAMP (98, 97, 54, and 4% transfer seen with 0, 1, 10, and 100 microM cAMP) and arachidonic acid (98, 73, 54, 31, and 0% dye transfer for 0.1, 1, 10, 100, and 1000 nM arachidonic acid) depressed dye transfer in cultured myocytes. Although the adenylate cyclase inhibitor 2',3'-dideoxyadenosine completely reversed forskolin-induced depression of dye transfer (1 microM forskolin, 22% transfer), it had no effect with lindane, indicating that lindane's depression of dye transfer was independent of adenylate cyclase activation. Lindane's inhibition of dye transfer was effectively reversed by growing myometrial cells under arachidonic acid-free conditions in the presence of eicosapentaenoic acid, a fatty acid that competes with arachidonic acid for the sn-1,2 position of membrane phospholipids: 0, 15, 40, and 88% dye transfer occurred in the presence of 0.01, 0.1, 1, and 10 microM eicosapentaenoic acid with 30 microM lindane. This implies that arachidonic acid release may be a critical event associated with lindane's inhibition of gap junctional communication in uterine myocytes.

Dde chlorinated insecticide 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (p,p'-DDD) increases intracellular calcium in rat myometrial smooth muscle cells.

A previous report from this laboratory showed that two DDT isomers, o,p'-DDT and p,p'-DDD, increased the frequency of spontaneous oscillatory contractions to a similar extent in isolated rat uterine segments. Because regulation of intracellular calcium is fundamental for the development of oscillatory contractions, the present study examined the effects of p,p'-DDD on intracellular free calcium concentration ([Ca2+]i) in individual rat myometrial smooth muscle cells loaded with the fluorescent Ca2+ indicator fura 2. In the presence of extracellular calcium, 50 and 100 microM p,p'-DDD significantly increased peak [Ca2+]i 586 and 921%, respectively, over basal [Ca2+]i. No significant effect was observed with 10 microM p,p'-DDD. In the absence of extracellular calcium, the response to 100 microM p,p'-DDD was significantly attenuated, with cells averaging a 108% increase in peak [Ca2+]i over basal levels, presumably through Ca2+ release from intracellular stores. Nifedipine and cadmium chloride, blockers of voltage-dependent calcium channels, inhibited 100 microM p,p'-DDD-stimulated increases in [Ca2+]i such that peak [Ca2+]i was increased 250% and 259%, respectively. Because of the prominent inhibition observed with the voltage-dependent calcium channel blockers, the effect of p,p'-DDD on membrane depolarization was examined using a cationic fluorescent indicator of membrane potential, [diS-C2(5)]. A concentration of 50 microM p,p'-DDD depolarized the cells by 35% of maximum during treatment with p,p'-DDD. The data demonstrate that p,p'-DDD increased [Ca2+]i in rat myometrial smooth muscle cells in a concentration-related manner, and that this increase was largely dependent on influx of extracellular calcium through dihydropyridine-sensitive, voltage-dependent calcium channels. The data further show that p,p'-DDD depolarized the plasma membrane, providing a possible mechanism for activation of voltage-dependent calcium channels. Additionally, another calcium source, perhaps an intracellular pool, contributes significantly less to the rise of [Ca2+]i. Whether p,p'-DDD initiates the calcium response by direct actions on the plasma membrane or by other means remains to be determined.

The role of gap junctional communication in contractile oscillations in arteries from normotensive and hypertensive rats.

OBJECTIVES: To test the hypotheses that vascular supersensitivity correlates with the appearance of contractile oscillations; vascular oscillations are mediated by gap junctions; and gap junctional communication is altered in the vasculature in stroke-prone spontaneously hypertensive (SHRSP) compared with Wistar-Kyoto (WKY) rats. DESIGN AND METHODS: Helical strips of mesenteric and tail arteries from SHRSP and WKY rats were mounted in tissue baths for measurement of isometric force. Cultures of mesenteric arterial cells were used for measurement of Lucifer yellow dye transfer and abundance of connexin43 mRNA, a monomer of gap junctions. RESULTS: Mesenteric arteries from SHRSP that displayed spontaneous oscillations were more sensitive to the contractile agonist 5-hydroxytryptamine than those from SHRSP and WKY rats that displayed no oscillations. In addition, SHRSP tail arteries displayed norepinephrine-induced oscillations. The putative gap junction up-regulator tetraethylammonium (10(-3)-10(-1) mol/l) induced oscillations (1-5 cycle/min) in arteries from both rat strains. These oscillations were not altered by endothelium removal or phentolamine and were blocked by heptanol (10(-3) mol/l). Although tetraethylammonium and heptanol caused similar effects in both arteries, heptanol-sensitive agonist-induced oscillations persisted only in the tail artery of SHRSP. Tetraethylammonium increased dye transfer between contiguous cells approximately 35% above basal levels both for SHRSP and WKY cells. In both cell lines, heptanol reduced basal- and tetraethylammonium-stimulated dye transfer. Total RNA from WKY rat and SHRSP cultured cells hybridized strongly and to a similar magnitude with a complementary DNA probe for messenger RNA for connexin43. CONCLUSIONS: Gap junctional communication is important in vascular reactivity and might play a part in the development of oscillations. Altered gap junctional communication could not be demonstrated in cell cultures nor in some contractile experiments. It is possible that the culture conditions failed to mimic conditions in vivo that differentially regulate gap junctions in the hypertensive state, but it is also possible that gap junctional activity is not abnormal in hypertension.

Lindane inhibition of gap junctional communication in myometrial myocytes is partially dependent on phosphoinositide-generated second messengers.

The ability of environmental contaminants to modulate gap junctional communication between uterine smooth muscle cells is generally unknown, despite recognition that myometrial gap junctions may play a role in synchronizing uterine contractions during parturition. The present study tested the hypothesis that the organochlorine pesticide lindane (gamma-hexachlorocyclohexane) inhibits gap junctional communication in myometrial myocytes due to the release of phosphoinositide-dependent second messengers. The effect on gap junctional communication by lindane was tested in cultured rat myometrial smooth muscle cells by monitoring transfer of the fluorescent dye Lucifer yellow. A rapid, concentration-dependent, but reversible inhibition of dye transfer was noted with 4-min exposures, and inhibition was complete with 10 microM lindane. Lindane also stimulated the production of the Ca(2+)-releasing species inositol 1,4,5-trisphosphate which peaked at 5 min (100 pmol/mg protein) and remained elevated after a 15-min exposure. To examine the possible inhibitory role of Ca2+ on gap junctions, the Ca2+ ionophore 4-br-A23187 was used. Although A23187 also inhibited gap junctional communication, inhibition was not complete even at concentrations that appeared cytotoxic (70% inhibition at 2 microM A23187). Cells were then loaded with the Ca2+ chelator BAPTA-AM, which blocked the lindane-induced rise in calcium, and dye transfer experiments with lindane were repeated in Ca(2+)-free medium. Inhibition of dye transfer was still complete under these conditions, showing that increased intracellular calcium was not required for lindane-induced inhibition of gap junctional communication. Subsequently, 10 microM lindane was shown to produce a sustained increase in protein kinase C (PKC) activity (31, 17, and 15 pmol of PKC peptide phosphorylated/min/mg protein for 2-, 5-, and 10-min exposures, respectively). Known activators of PKC, 12-O-tetradecanoylphorbol 13-acetate (TPA) and 1,2-dioctanoyl-sn-glycerol, abolished gap junctional communication at nanomolar concentrations. Although use of the PKC inhibitor staurosporine failed to reverse lindane's inhibitory action, depletion of PKC activity through prolonged exposure to TPA partially reversed lindane's effect. This suggests that PKC activation potentiates but does not solely mediate lindane's inhibitory action on gap junctional communication.

Gap junctional communication and vascular smooth muscle reactivity: use of tetraethylammonium chloride.

Oscillatory contractions in uterine smooth muscle are mechanistically related to gap junction complex formation. We have tested the hypothesis that agonist-induced oscillations in vascular smooth muscle are also mediated by gap junctions and that gap junctions are important for vascular smooth muscle cell communication. Total RNA from cultured Wistar-Kyoto rat (WKY) mesenteric arterial cells hybridized strongly with a cDNA probe for the message for connexin43, a monomer of the gap junction. In these same cells, the quaternary ion tetraethylammonium (TEA) (10 mM) increased Lucifer yellow dye transfer between contiguous cells, a measure of cell-to-cell communication via gap junctions, approximately 35% above basal levels. Heptanol, an established inhibitor of gap junction communication, completely blocked both basal- and TEA-stimulated dye transfer between neighboring cells. In other experiments, helical strips of superior mesenteric and tail arteries from WKY rats were mounted in tissue baths for measurement of isometric contractile force. TEA (10(-3)-10(-1) M) induced oscillatory contractions (1-5 cycle/min) in both mesenteric and tail arteries. Removal of endothelium did not affect the pattern of TEA-stimulated oscillations. Oscillations to TEA were blocked in a concentration-dependent manner in both arteries by heptanol (10(-7)-10(-3) M). Heptanol (10(-3) M) also significantly reduced (40%) acetylcholine-induced relaxation in the mesenteric artery (contracted with phenylephrine).(ABSTRACT TRUNCATED AT 250 WORDS)

Female reproductive and developmental toxicology: overview and current approaches.

In recent years, concern about possible female reproductive and developmental toxicity due to environmental contaminants, such as PCBs, has been growing. Because this area of toxicology had not been emphasized prior to this time, there are many gaps in current knowledge about female developmental and reproductive toxicology and only a limited number of validated tests to assay effects of toxicants on various parts of the reproductive and developmental cycle. This article reviews the current state of knowledge on this topic and also explores a variety of techniques for assessing female reproductive and developmental toxicity. These include an assay of the state of intercellular communication among the embryo, fetus and placenta; protocols for assessing toxicity in early pregnancy; and techniques for evaluating the role of glutathione in protecting the conceptus from xenobiotics.